High dose Ara-C (HIDAC) is a highly effective drug against human AML, but resistance of AML blast progenitor cells to HIDAC leads to eventual relapse and treatment failure. Ara-C DNA incorporation causes inhibition of nascent DNA chain elongation and DNA strand breaks, which triggers the mitochondrial (intrinsic) pathway of apoptosis. The antiapoptotic Bcl-2 or Bcl-xL inhibits, while the proapoptotic Bax promotes HIDAC-induced mitochondrial events of apoptosis. RAD9, a human protein involved in the control of a cell cycle checkpoint, is phosphorylated following DNA damage and interacts with Bcl-2 and promotes apoptosis. Following DNA damage, the cytosolic Bax undergoes a conformational change, translocates to the mitochondria and interacts with Bc1-2 or Bcl-xL to promote apoptosis. However, the role of RAD9 or Bax in sensing and linking HIDAC-induced DNA damage to the initiation of the mitochondrial pathway of apoptosis of human AML blast progenitor cells has yet to be determined. HIDAC treatment upregulates the death receptor DR5 and sensitizes cultured human AML cells to the extrinsic, Apo-2L/TRAIL-induced pathway of apoptosis. But, the mechanism and regulation of HIDAC-induced sensitization to Apo-2L/TRAIL needs to be further investigated. Finally, during HIDAC-induced common pathway of apoptosis, the inhibitory control of the IAP family of proteins on the activity of the effector caspases, or its abrogation by Smac (second mitochondrial activator of caspases) remains to be determined in AML blast progenitor cells. Importantly, by comparing their cytotoxic effects against normal human bone marrow progenitor cells the anti-AML selectivity of these strategies needs to be evaluated. Therefore, the specific aims of this proposal are: AIM 1: To determine whether HIDAC-induced DNA damage induces Rad9 phosphorylation and/or Bax conformational change, promotes their translocation to the mitochondria, inactivates Bc1-2 and Bc1-xL and initiates the mitochondrial events leading to apoptosis of AML blast progenitor cells. AIM 2: To determine the mechanism by which HIDAC potentiates Apo-2L/TRAIL-induced DISC, as well as to determine whether abrogation of the survival signalings of NFkB and Akt kinase would further improve HIDAC-enhanced assembly and activity of Apo2L/TRAIL-induced DISC and apoptosis of AML blast progenitor cells. AIM 3: To determine the antiapoptotic role of IAP family members as well as the potentiating effect of Smac or other anti-IAP strategies on HIDAC-induced apoptosis of purified human AML blast progenitor cells. AIM 4: To compare the effects of combinations of HIDAC and Apo-2L/TRAIL and/or Smac peptide on the clonogenic survival and apoptosis of AML blast progenitor versus CD34+ normal bone marrow progenitor cells. Investigation into these diverse mechanisms that may regulate HIDAC-triggered apoptotic signaling can potentially define novel therapeutic strategies to potentiate HIDAC activity against human AML.